My laboratory is fascinated by four questions: How do membrane-binding domains interact with membranes to target signal transduction proteins to various subcellular compartments? How can pH-induced refolding on membranes of diphtheria toxin T-domain and colicins cause highly charged helices to cross lipid bilayers? How can short, highly basic "Trojan" peptides - such as penetratin and HIV-TAT peptide - cross pure lipid vesicles unaided, as some claim? How do simple toxins and antimicrobial peptides permeabilize membranes? The shared theme of these challenging questions is mediation of biological function through direct physicochemical interactions of proteins with lipid bilayers, especially the bilayer interface: Subcellular specificity of signal transduction domains is expressed directly through interfacial interactions. Breaching of the lipid bilayer by diphtheria toxin, Trojan peptides, and antimicrobial peptides begins with interfacial interactions, which 'set up' the peptide-bilayer complex for protein insertion/translocation. The physical principles underlying these interactions also underlie membrane protein (MP) stability, and are the key to predicting 3D structure from sequence. Our unique capabilities for combined structural and thermodynamic studies of peptide-bilayer interactions have brought us to the threshold of significant new advances, not only in fundamental principles, but also in their application to challenging biological problems. Our specific aims embrace the broad objectives of enlarging our understanding of basic physicochemical principles, developing new structural methods for thermally disordered membranes, and applying these principles and methods to important biological problems: (1) Clarify how hydrophobic and electrostatic interactions work together at membrane interfaces to mediate protein-lipid interactions. (2) Advance the development of a novel diffraction method - Molecular Dynamics/Absolute Scale (MoDAS) refinement- in order to gain dynamic structural images of peptides interacting at the atomic level with thermally disordered lipid bilayers. (3) Elucidate the mechanism of pH-induced refolding of diphtheria toxin T-domain on membranes in order to understand how the protein translocates its catalytic domain across endosomal membranes. (4) Determine if so-called "Trojan" peptides - such as penetratin and TAT - can cross pure lipid bilayers unaided, and if so, under what conditions. (5) Expand and improve physicochemical rules for predicting the binding and folding of peptides at membrane interfaces, and establish a structural context for them.